This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In previous work on channel formation using spin labeled Gramicidin A (GAsl) we found that the non-channel form prevails in the gel phase of mismatching lipids (DPPC, DSPC). We have now addressed the assignment of non-channel forms and studied the complex equilibrium in the gramicidin/lipid system, which includes channels (i.e. head-to-head dimers [HHD]), non-channel dimers, and monomers. To study non-channel forms of spin labeled GA, which can aggregate, we labeled two different sites on the same gramicidin molecule, instead of measuring the distance between spin labels attached at the same position on both dimer-forming molecules. This intramolecular distance between the spin labels could be used as a fingerprint of the particular conformation. Interactions between spin labels on different gramicidin molecules were eliminated by substantially diluting the double spin-labeled gramicidin by unlabeled gramicidin. Several new spin labeled gramicidin compounds were synthesized and used. In octanol, gramicidin exists mainly as a double helical (DH) left-handed antiparallel dimer, and we determined interspin distances for this structure. GAD with both N- and C-termini labeled and GALN, single-labeled at the N-terminus, substantially impair their propensity to HHD formation. In most lipids we observed two different conformations of GAD. The interspin distance in the main fraction (20.0[unreadable]) is consistent with the monomeric state. Double helices (31.6[unreadable], exactly as in octanol) are a minor component with the fraction changing in the saturated lipid series in the following order: DLPC<DMPC<DSPC<DPPC. We also showed by DQC that both gramicidin monomers and DH exist in all studied lipid phases only in aggregates.